The proposed studies are designed to investigate the regulation of phosphoinositide breakdown by hormones in a cell-free system from the blowfly salivary gland. Phosphoinositide breakdown has been linked to a receptor mechanism, distinct from the cyclic AMP system, involved in the elevation of cytosolic Ca2+. The system to be used in these studies, the salivary gland of the blowfly, Calliphora erythrocephala, selectively incorporates (3H)ubisutik and (32P)Pi into the pool of phosphoinositides involved in hormone action. Using a membrane fraction, obtained from salivary glands prelabeled with (3H)inositol, it is possible to demonstrate 5-hydroxytryptamine-stimulated production of inositol phosphates derived from phosphoinositide breakdown. Hormone-stimulated breakdown in the cell-free system is rapid and is mediated through a phospholipase active at physiological pH. The relationship between phosphatidylinositol and the polyphosphoinositides in the cell-free system has not been defined. The isolated membrane fraction will be used for the biochemical characterization of the phospholipases involved in hormone-stimulated phosphoinositide breakdown, the mechanism by which hormone stimulates phosphoinositide breakdown, the nature of the active pool of phosphoinositides involved in hormone action and the metabolic interrelationship between phosphatidylinositol and the polyphosphoinositides.